Our laboratory is studying the 70-Kda heat shock proteins which act as molecular chaperones, that is disaggregate, fold, and unfold proteins in numerous processes in vivo. We have taken several approaches to understanding the mechanism of action of these proteins. First, we are carrying out a detailed investigation of the mechanism of action of the bovine brain uncoating ATPase, a constitutive 70-Kda protein, in uncoating bovine brain clathrin-coated vesicles with particular emphasis on the relationship between ATPase activity and uncoating; the uncoating process is, by far, the best defined biochemical system for studying the action of a 70-Kda protein. In carrying out this study we have discovered that a 100-Kda protein cofactor is required for the uncoating process to occur. The activity of this cofactor appears to saturate at a 1 to 10 ratio to both clathrin and the uncoating ATPase. We have also discovered a new 20-Kda assembly protein which induces the formation of normal clathrin baskets. This assembly protein, which occurs in large amounts in brain extracts, has a much lower molecular weight than other previously discovered assembly proteins. We are also studying the role of the bound nucleotide in the binding of both clathrin and other peptide substrates to the uncoating ATPase. We previously showed that clathrin and several peptide substrates bind to the same site on the uncoating ATPase. Our current studies show that while dissociation of both clathrin and the peptides is rapid in the presence of ATP; bound ADP and Pi decrease their rates of dissociation by several orders of magnitude. These data suggest that ATP binding rather than ATP hydrolysis may be responsible for dissociation of substrates from 70-Kda proteins. Using nucleotide free enzyme, we have also been able to demonstrate that the effects of ATP and ADP are very specific. Nucleotide analogues like AMP- PNP and dATP have no effect on the properties of the nucleotide-free enzyme possibly because they bind 2 to 3 orders of magnitude weaker than ATP and ADP. Finally, we have cloned the bovine brain uncoating ATPase and have succeeded in expressing it in large amounts in E Coli as a GST- fusion protein. We plan to study the effect of site-directed mutagenesis on the properties of this protein.